Enzymatic synthesis of phosphorothioate oligonucleotides using restriction endonucleases

ABSTRACT

The present invention relates to methods of synthesizing phosphorothioate oligonucleotides. In particular, it relates to the use of certain restriction endonucleases to cleave phosphorothioate oligonucleotides which contain restriction endonuclease recognition sequences. These restriction sequences facilitate the cleavage of relatively cleavage resistant phosphorothioate oligonucleotides thus facilitating their separation and purification after synthesis.

FIELD OF THE INVENTION

The present invention relates to methods of synthesizingphosphorothioate oligonucleotides. In particular, it relates to the useof certain restriction endonucleases to cleave phosphorothioate nucleicacids which contain restriction endonuclease recognition sequences.

BACKGROUND OF THE INVENTION

Nucleic acids are linear polymers consisting of individual nucleotidesubunits which are covalently linked together via phosphodiester bonds.Oligonucleotides are now widely used in the biomedical field as nucleicacid sequencing primers, diagnostic probes and modulators of genefunction. One of the most promising uses of oligonucleotides is in thefield of antisense therapeutics.

Oligonucleotides can be conveniently synthesized using enzymatic orchemical methods, with the latter generally providing for larger scaleproduction than the former. One of the most widely used methods ofchemically synthesizing oligonucleotides is based on phosphoramiditesolid-phase chemistry (See Methods in Molecular Biology, Volume 20:Protocols for Oligonucleotides and Analogs, S. Agrawal editor, HumanaPress inc., Totowa, N.J., 437-463 (1993)) Now fully automated, thismethod can be used to chemically produce oligonucleotides in commercialquantities. (See Oligonucleotides and Analogues: A Practical Approach.Edited by F. Eckstein, I.R.L. Press, Oxford, England, 1-24 (1991).)

Despite the convenience of chemical synthesis, the number of steps andthe harshness of the chemicals involved leads to the formation of aproduct which may contain toxic chemical impurities, such as damagednucleotide bases. Because the level of impurities is normally relativelylow, chemically synthesized oligonucleotides are still suitable for usein many different applications. However, certain applications require aproduct which is substantially free of chemical impurities. Inparticular, when the application involves a biological system, thepresence of chemical impurities can have a deleterious effect. Moreover,when the application involves an in vivo therapeutic agent, chemicalpurity is essential. Enzymatic synthesis, which can be used to producean oligonucleotide product in an aqueous solution that is essentiallyfree of toxic chemical impurities and hazardous byproducts is thuspreferred for these applications.

The use of oligonucleotides in biological systems is also compromised bythe presence of nucleases which catalyze the breakdown of nucleic acidsby hydrolysis of phosphodiester bonds (See The Biochemistry of theNucleic Acids: Chapter 4, Degradation and Modification of Nucleic Acids,Roger L. P. Adams et al., Chapman a Hall, London, England, 97-108(1992)). Such a breakdown can cause a significant reduction in thebiological activity of oligonucleotides in vivo thus resulting indiminished therapeutic effectiveness. This degradation can be controlledby modifying or substituting the phosphodiester bonds with a morenuclease-resistant analog, such as phosphotriester, phosphorothioate ormethylphosphonate.

Phosphorothioate-containing oligonucleotides efficiently resistdegradation by many nucleases, and are thus preferred for use in somebiological systems. Phosphorothioate linkages have a sulfur in place ofoxygen as one of the non-bridging atoms bonded to phosphorous. Thissubstitution produces chirality at the phosphorous which is designatedas having either the Rp or Sp diastereomer orientation. Since the chiralorientation is an important factor which influences duplex structure,enzyme recognition, conformation and/or hybridization kinetics, it isdesirable to use chirally pure phosphorothioate-containingoligonucleotides. Other modified oligonucleotides such as thosecontaining phosphotriester and methylphosphonate linkages also contain asubstitution of one of the oxygen atoms bonded to phosphorous and thusexist as diastereomers.

Chemical synthesis of phosphorothioate-containing oligonucleotidesgenerally lacks stereoselectivity and results in the formation of aproduct which is a heterogeneous mixture of two different chiralspecies. Attempts to stereochemically control the synthesis of chirallypure oligonucleotides have met with mixed success (Stec, et al., NucleicAcids Research, 19(21): 5883-5888 (1991)). Cook (U.S. Pat. No.5,212,295) has described the chemical synthesis of modifiedoligonucleotides with greater than 75% chiral purity. In comparison,enzymatic synthesis of phosphorothioate-containing oligonucleotides canbe used to produce chirally pure product, since several polymerases formonly phosphorothioate linkages having the Rp orientation (Eckstein, Ann.Rev. Biochem. 54: 367-402 (1985)).

The enzymatic synthesis of other modified oligonucleotides is also wellknown in the art. Methylphosphonate-containing oligonucleotides can beproduced enzymatically using DNA polymerases α and ε from humanplacenta, DNA polymerase β from rat liver, and reverse transcriptasesfrom HIV and arian myeloblastosis virus (Dyatkina, et al., Nucleic AcidsResearch Symposium Series 24: 238 (1991)).

The economical enzymatic synthesis of any oligonucleotide, whethermodified or not, depends on the ability to efficiently utilize thecomponents of the synthesis reaction to form a product which issufficiently pure for its intended use. In some instances, it isdesirable to use synthesis components which are capable of functioningrepeatedly and are thus "reusable". For example, Richards et al. (PCT WO92/05287) describes the use of reusable synthesis templates whichfunction repeatedly in the same synthesis reaction. In other instances,it may be desirable to use synthesis components which function only oncein a synthesis reaction and are thereafter degraded or renderednonfunctioning. Walder, et al. (European Patent Application 496,483 A2)describe the use of RNA-containing primers that are cleaved in order toprevent the formation of undesired amplification products in subsequentsynthesis reactions.

Even though many of the prior art methods are suitable for use in theenzymatic synthesis of oligonucleotides, large scale synthesis ofchirally pure oligonucleotides has yet to be optimized. It is thereforean object of the present invention to provide for the economicalsynthesis of oligonucleotides which are chirally pure.

Regardless of whether phosphorothioate-containing oligonucleotides arechemically or enzymatically synthesized, their resistance to hydrolysismakes them difficult to cleave as is sometimes desirable duringsynthesis and/or purification. Most studies involvingphosphorothioate-containing oligonucleotides and restrictionendonucleases have centered on the design of oligonucleotides which areprotected from cleavage (Nakamaye, et al., Nucleic Acid Research 14(24):9679-9699 (1986); Richards et al., supra; and Vosberg, et al., journalof Biol. Chem., 257(11): 6595-6599(1982)).

The present invention involves the identification of several restrictionendonuclease recognition sequences ("restriction sequences") which arecapable of being recognized and cleaved by restriction endonucleases,even when present in fully phosphorothioated oligonucleotides. In theenzymatic synthesis of phosphorothioate oligonucleotides, utilizingtemplates which direct the synthesis of products containing theserestriction sequences facilitates separation and purification ofoligonucleotides.

None of the references herein are admitted to be prior art.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of several restrictionendonuclease recognition sequences ("restriction sequences") which arecapable of being recognized and cleaved by certain restrictionendonucleases even when present in fully phosphorothioatedoligonucleotides ("phosphorothioate oligonucleotides".) Accordingly, thepresent invention involves a method of synthesizing at least onephosphorothioate oligonucleotide comprising the steps of (a) providing anucleic acid template, the template comprising a primer binding regionand an oligonucleotide complementary region; (b contacting the templatewith a nucleic acid primer able to hybridize to the template to form atemplate-primer hybrid; (c) incubating the template-primer hybrid in thepresence of at least one DNA polymerase under conditions in which DNAsynthesis occurs to form a primer extension product, wherein the primerextension product comprises at least one restriction endonucleaserecognition sequence selected from the group consisting of 5'-GGCC-3',5'-GCGC-3', 5'-CCNGG-3', 5'-TCGA-3'; and (d) cleaving the primerextension product to separate the primer and the oligonucleotide or theoligonucleotides and, if necessary, to separate the oligonucleotidesfrom each other, wherein at least one cleavage is accomplished bycontacting the primer extension product with an effective amount of arestriction endonuclease capable of recognizing the restrictionendonuclease recognition sequence selected from the group consisting ofHae III, HinP I, ScrF I, and Taq.sup.α I.

The template can be either DNA or RNA, but is preferably RNA. Eachtemplate is composed of two regions: a primer binding region (or aself-complementary region in the case of a self-priming template), andan oligonucleotide complementary region. The oligonucleotidecomplementary region may contain two or more subregions, each of whichcan have the same or a different nucleic acid sequence, such that eachsubregion directs the synthesis of an individual oligonucleotide. Inthis manner, oligonucleotides having the same or different lengthsand/or sequences may be formed from the same template.

The primers form template-primer hybrids when hybridized to thetemplate. Alternatively, a separate primer need not be supplied if aself-priming template is used and the 3'-terminus of the self-primingtemplate serves as the primer. Primers which are particularly usefuleither contain or form at least one cleavable nucleotide linkage which,when incorporated into the primer extension product, can serve as aconvenient cleavage site for separation of the primer from theoligonucleotide or oligonucleotides (i.e. the "oligonucleotideproduct").

The template-primer hybrid, or the self-priming template, directs thesynthesis of a primer extension product via sequential addition ofnucleotides or modified nucleotides. The use of modified nucleotides ispreferred for formation of modified oligonucleotides which arenuclease-resistant.

Cleavage of the primer extension product to separate the primer from theoligonucleotide product can be facilitated by using a primer whichcontains or forms at least one cleavable nucleotide linkage. Such aprimer is, for example, a 3'-ribonucleotide primer which forms aclearable linkage which is susceptible to alkaline hydrolysis or RNasecleavage. Alternatively, when a primer is used which is DNA with asingle deoxyuridine residue at its 3'-terminus, the primer extensionproduct can be cleaved by excising the deoxyuridine residue using acombination of DNA glycosylase, AP-endonuclease and exonuclease.

Additionally, when the oligonucleotide complementary region containsmore than one subregion, incorporation of restriction sequences into theprimer extension product is used to position cleavage sites in theoligonucleotide product to facilitate separation of the individualoligonucleotides. Cleavage to separate the individual oligonucleotidesfrom each other can then be accomplished using the appropriaterestriction endonuclease.

When digestion of the template is used to separate the primer extensionproduct from the template, cleavage (to separate the primer from theoligonucleotide product and the individual oligonucleotides from eachother) can take place before, during or after template digestion.Digestion of an RNA template can be accomplished via alkaline hydrolysisand/or an appropriate Rnase. Digestion of a DNA template can beaccomplished by using an appropriate DNase.

BRIEF DESCRIPTION OF THE DRAWINGS

As used in the Figures described below, the abbreviations will have thefollowing meaning:

pbr=primer binding region

ocr=oligonucleotide complementary region

sr=subregion

cs=cleavage site

scr=self-complementary region

R=ribonucleotide

FIG. 1 illustrates several different template structures, eachcontaining a primer binding region and an oligonucleotide complementaryregion.

FIG. 2 illustrates several different template structures, eachcontaining a primer binding region, and an oligonucleotide complementaryregion which consists of three separate subregions.

FIG. 3 illustrates the use of a template precursor which is cleaved toform two separate templates.

FIG. 4 illustrates the enzymatic synthesis of an oligonucleotide using aself-priming template.

FIG. 5 illustrates the enzymatic synthesis of an oligonucleotide using acombined primer/template.

FIG. 6 illustrates the enzymatic synthesis of an oligonucleotide using a3'-ribonucleotide primer.

FIG. 7 illustrates the enzymatic synthesis of two or moreoligonucleotides from a single template using a primer-extension productcontaining more than one type of cleavage site, one being the phosphatelinkage 3' to a ribonucleotide residue which was introduced into theprimer extension product by using a 3'-ribonucleotide primer, and theother being a restriction endonuclease recognition sequence.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention concerns the identification of restrictionendonuclease recognition sequences, and the synthesis and cleavage ofphosphorothioate oligonucleotides which contain these sequences. Inorder to more clearly describe the subject matter of the presentinvention, certain terms used herein shall be defined as follows unlessotherwise indicated:

Chirally pure: "Chirally pure", when used to refer to aphosphorothioate-containing oligonucleotide, means that theoligonucleotide contains only Rp or Sp diastereomers.

Cleavage Site:

"Cleavage site" means a location in a nucleic acid that is susceptibleto hydrolysis of at least one native or modified phosphodiester bond inthe sugar-phosphate backbone of the nucleic acid. When used to refer toa restriction endonuclease recognition sequence, the term "cleavagesite" means the clearable linkage (denoted as "/") in a sequence ofnucleotides which are specifically recognized by a restrictionendonuclease which binds to the sequence to effectuate cleavage. Forexample, the sequence GGCC is recognized by Hae III and cleavage occursat GG/CC. "Cleavage site", as used herein, can also mean the linkage 3'to a ribonucleotide residue, which is susceptible to cleavage by anagent such as RNase or alkali.

Cleave:

"Cleave" means to cause a break in the sugar-phosphate backbone of anoligonucleotide using an endonuclease or other cutting agent.

Complementary:

"Complementary", when used to refer to a nucleic acid, means a nucleicacid of one polarity containing a sequence of nucleotides whose basespair with the nucleotide bases of another nucleic acid of oppositepolarity, i.e. adenine ("A") pairs with thymine ("T") or uracil ("U"),and guanine ("G") pairs with cytosine ("C"). For example, a nucleic acidhaving the sequence GCAU in the 5' to 3' direction is "complementary" toa nucleic acid having the sequence CGTA in the 3' to 5' direction. Useof the term complementary herein is intended to include those nucleicacids which are substantially complementary. Complementary nucleic acidscan also be referred to as one being the plus ("(+)") or "sense" strandand the other being the minus ("(-)") or "antisense" strand.

Diastereomer:

"Diastereomer" means a chiral compound that can exist as two differentchiral species which, in the case of phosphorothioate-containingoligonucleotides, depends on the orientation of sulfur in relation tothe chiral phosphorous. The two possible orientations are Rp and Sp,thus the chiral species are referred to as being Rp or Sp diastereomers.

Digestion:

"Digestion" means the degradation of a nucleic acid into its individualnucleotides (complete digestion) or into short segments (partialdigestion).

dNTP:

"dNTP" means a deoxynucleoside triphosphate, wherein N refers to thenucleotide bases, i.e. dATP means deoxyadenosine triphosphate, dCTPmeans deoxycytosine triphosphate, etc. The term "dNTP", or "nucleotide",is intended to include an unmodified deoxynucleoside triphosphate, aswell as a deoxynucleoside triphosphate with a modified α linkage, suchas deoxynucleoside triphosphorothioate and deoxynucleosidemethylphosphonate.

dNTPαS:

"dNTPαS" means a deoxynucleoside triphosphates having a sulfur atomsubstituted for oxygen at the alpha phosphorous position wherein N isdefined as described for dNTP.

Hairpin:

"Hairpin" means the looped self-hybridized structure that a self-primingtemplate takes when the two intra-complementary regions, which areseparated by a non-complementary region that becomes the "loop", form aduplex. See FIG. 4.

Hybridization:

"Hybridization" means the formation of a stable duplex betweennucleotide sequences that are substantially complementary.

Modified:

"Modified", when used to refer to a nucleic acid, means a nucleic acidin which any of the natural structures have been altered. These includethe native phosphodiester linkages (also referred to herein as simply"phosphodiester linkages"), the sugars (ribose in the case of RNA ordeoxyribose in the case of DNA) and/or the purine or pyrimidine bases.Modified phosphodiester linkages include phosphorothioates,phosphotriesters, methylphosphonates and phosphorodithioates.

Nucleic Acid Sequence:

"Nucleic acid sequence", or "sequence", means both a nucleic acid havinga particular sequence of nucleotides, and also the sequence or order ofnucleotides present in a particular nucleic acid. Which of these twomeanings applies will be apparent from the context in which this term isused.

Oligonucleotide:

"Oligonucleotide" means a relatively short segment of a nucleic acidpolymer. Although a limitation to any preise length is not intended,oligonucleotides are generally between 8 to 100 nucleotides in length.

Polarity:

"Polarity" means the orientation of a nucleic acid polymer which iscreated when the C3 position of one deoxyribose (or ribose) moiety islinked together with the C5 of the adjacent deoxyribose (or ribose)moiety via a native or modified phosphodiester linkage. Polarity iscreated by the sequence of bases relative to the 5' and 3' ends. Twocomplementary strands are of "opposite polarity" when the sequence ofbases read from the 5' to 3' direction of one strand and the sequence ofbases read from the 3' to 5' direction of the other give correspondingWatson and Crick base paris at each position.

Phosphorothioate Oligonucleotide:

"Phosphorothioate oligonucleotide" means an oligonucleotide having allphosphorothioate linkages (also referred to as a "fullyphosphorothioated oligonucleotide").

Phosphorothioate-containing oligonucleotide:

"Phosphorothioate-containing oligonucleotide" means an oligonucleotidehaving at least one but less than all phosphorothioate linkages (alsoreferred to as a "partially phosphorothioated oligonucleotide").

Polymerase:

"Polymerase" means an enzyme which is capable of catalyzing thesynthesis of a complementary copy of a nucleic acid template, which inthe case of a DNA polymerase is brought about by the sequential additionof deoxynucleotides to a primer.

Primer:

"Primer" means an oligonucleotide that is complementary to a templatethat hybridizes with the template to give a template-primer hybrid forinitiation of synthesis by a DNA polymerase, such as reversetranscriptase or bacteriophage T4 DNA polymerase, and which is extendedby the sequential addition of covalently bonded nucleotides linked toits 3' end that are complementary to the template. The result is theformation of a primer extension product.

RNase H:

"RNase H" means an enzyme that degrades the RNA strand of an RNA:DNAduplex.

Self-priming template:

"Self-priming template" means a template which is capable of initiatingsynthesis by a polymerase without the addition of a separate primer. The3'-terminus of a self-priming template serves as the primer.

Self-Priming Template Extension Product:

"Self-priming template extension product" means the product which isformed during oligonucleotide synthesis using a self-priming template.As used herein, a self-priming template extension product is a type ofprimer extension product to which the template remains covalentlyattached after synthesis.

Substantially Complementary:

"Substantially Complementary", when used to refer to a nucleic acid,means having a sequence such that not all of the nucleotides exhibitbase pairing with the nucleotides of another nucleic acid, but the twonucleic acids are nonetheless capable of forming a stable hybrid underspecified conditions.

Template:

"Template" means a nucleic acid molecule that is able to be copied by apolymerase, and which has a sequence of nucleotides which will providethe pattern and serve as substrate for producing a desiredoligonucleotide. In order to serve as such, the template must contain asequence which is capable of hybridizing with a primer (a "primerbinding region"). A self-priming template must contain a sequence whichis capable of forming a hairpin structure (a "self-complementaryregion").

Template precursor:

"Template precursor" means an oligonucleotide which contains at leastone copy of the template nucleic acid sequence and can be cleaved toproduce at least one template nucleic acid.

Template-primer hybrid:

"Template-primer hybrid" means a partially double-stranded nucleic acidwhich is formed when a template and a primer hybridize.

Oligonucleotides have many different uses in biomedical science. To namea few, they can be used as labelled probes for detection of specificnucleic acid sequences in diagnostic assays (Kohne et al., U.S. Pat.Nos. 4,851,330 and 5,288,611); they can be used to promote formation ofregions in an RNA target which are accessible to hybridization oflabelled probes (See Hogan et al., U.S. Pat. No. 5,030,557); they can beused as investigational tools for the sequencing of DNA (Biggin et al.,P.N.A.S. 80: 3936-3965 (1983); and, especially in the case of modifiedoligonucleotides, they can be used in biological systems. Because of theability of certain modified nucleic acids to resist degradation bynucleases, they are much more stable in such systems than theirunmodified (native) phosphodiester counterparts. This makes them idealfor use in vivo as antisense therapeutic agents.

"Antisense" refers to the use of oligonucleotides as regulators of genefunction. An antisense oligonucleotide, i.e. an oligonucleotide having anucleic acid sequence which is complementary to that of the "sense"nucleic acid to which it is targeted, can function in many differentways to modulate gene function. When the targeted nucleic acid ismessenger RNA ("mRNA"), it may function by preventing translation of themRNA into protein or inhibiting binding of ribosomes. When the targetednucleic acid is DNA, it may prevent transcription into mRNA.

The use of phosphorothioate-containing oligonucleotides in the field ofantisense therapeutics is now widespread. Examples of a few of theapplications are: the treatment of acute myeloblastic leukemia (Bergot,et al., Nucleic Acids Symposium Series No. 29, pg. 57 (1993)); theinhibition of HIV replication (Matsukura et al., Gene 72: 343-347(1988)); and the inhibition of influenza virus replication (Leiter etal., P.N.A.S. 87: 3430-3434 (1990) and Cohen et al., U.S. Pat. Nos.5,264,423; 5,276,019; and 5,286,717).

Oligonucleotides which are useful for particular purposes can be"designed" to hybridize with selected portions of a target sequence and,by varying such things as length and target binding region, theirsuitability for certain purposes can be maximized. For example, if thedesired oligonucleotide product is an antisense therapeutic to be usedin targeting the protein coding region of mRNA, an oligonucleotide mightbe chosen which would be complementary to an accessible region of themRNA sequence, i.e. one expected not to contain secondary structure,with sufficient length and complementarity to a unique sequence so as tonot exhibit cross-reactivity with non-targeted nucleic acids.

Although appropriate oligonucleotide length depends entirely on theparticular use for which the oligonucleotide is designed, certaingeneralizations are possible. If the oligonucleotide is to be used as alinker in cloning, it will preferably be between about 6 and 60nucleotides in length. If it is to be used as a sequence-specifichybridization probe, it will preferably be between 12 and 60 nucleotidesin length. If it is to be used as a polymerase chain reaction ("PCR")primer, it will preferably be between about 8 and 35 bases. If it is tobe used as a ligase chain reaction ("LCR") primer, it will preferably bebetween about 8 and 35 nucleotides in length. If it is to be used as anantisense therapeutic agent, it will preferably be between about 12 and50, and more preferably between about 15 and 30 nucleotides in length.

The present invention is particularly well-suited for use inenzymatically synthesizing phosphorothioate oligonucleotides which arechirally pure. It generally involves the use of templates and primers toform template-primer hybrids (or the use of self-priming templates toform hairpins), the synthesis of primer extension products, and thecleavage and separation of the desired individual oligonucleotides. Eachof these aspects are more fully described below.

The Template

The template can be either DNA or RNA, or modified DNA or RNA, but ispreferably unmodified RNA to facilitate digestion. Templates having aspecific nucleic acid sequence can be prepared using any known chemicalor enzymatic methods. In addition, templates can be prepared using anyknown recombination or cloning methods, such as with plasmids, M13 DNA,etc. Chemical synthesis can be conveniently performed according to themethod described by Stec et al. (J. Am. Chem. Soc. 106: 6077-6079(1984)) using the phosphoroamidite method and an automated synthesizer,such as Model 380-B (Applied Biosystems, Inc., Foster City, Calif.).

Particularly useful methods for synthesizing templates are based onenzymatic amplification technologies for producing multiple copies fromas few as a single copy of a nucleic acid. The following references,which are incorporated herein in their entirety, are representative ofseveral different amplification procedures which can be employed forproduction of template. PCR for the production of DNA is described byMullis, et al. (See U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159,and Methods in Enzymology, Volume 155, 1987, pp. 335-350.) A Qβreplicase-based amplification system for producing RNA is described byKramer, et al., in U.S. Pat. No. 4,786,600.

Because single-stranded template preparation lacking complementarystrands are ideal, preferred amplification procedures utilizetranscription-based amplification systems, which produce multiple copiesof single-stranded RNA from double-stranded DNA. (See Burg, et al., WO89/01050 and Gingeras, et al., WO 88/10315.) Still othertranscription-based amplification methods are described by Kacian, etal. (WO 91/01384 and WO 93/22461) and McDonough, et al., (WO 94/03472).PCR has also been used to produce single-stranded RNA from doublestranded DNA from which RNA is synthesized in vitro using DNA-directedRNA polymerase. (See Murakawa, et al., DNA 7:287-295 (1988)).

The nucleic acid sequence of a template will necessarily have at leasttwo nucleic acid subsequences; one of which is complementary to theprimer, i.e. the "primer binding region", and another of which iscomplementary to the oligonucleotide product to be synthesized, i.e. the"oligonucleotide complementary region". In addition, the template can bein the form of a plasmid, or a PCR product (after denaturation andstrand separation), or restriction fragments or linearized versionsthereof. For examples of different template structures, see FIG. 1.

In order to use a single template for the production of more than oneoligonucleotide, a template is synthesized which contains anoligonucleotide complementary region having more than one subregion,such that each subregion is complementary to and capable of directingthe synthesis of an individual oligonucleotide. See FIG. 2. Using such atemplate, the oligonucleotide complementary region is complementary toand directs the synthesis of an "oligonucleotide product", which canthen be cleaved after synthesis to separate the individualoligonucleotides.

A template with more than one such subregion must also be designed tofacilitate separation of the individual oligonucleotides from each otherafter synthesis. This can be accomplished by incorporating restrictionsequences into the primer extension product which are specificallypositioned to allow for cleavage to separate the individualoligonucleotides. (See Cleavage of the Primer-Extension Product belowfor a more complete description of cleavage to separate individualoligonucleotides.)

The individual oligonucleotides synthesized in this manner may have thesame or different sequences, as well as the same or different lengths.This method is very useful when pairs of oligonucleotides which do notrequire further separation are to be used for a particular application.

The template can be formed such that it is ready for use in thesynthesis of oligonucleotides, or it can be formed as a templateprecursor which can then be converted to a usable template by cleavagewith an endonuclease or other such cleavage mechanism. (See Cleavage ofthe Primer-Extension Product below for a complete description ofcleavage mechanisms.) See FIG. 3.

Additionally, templates can be self-priming, i.e. capable of initiatingsynthesis without the need for a separate primer. Instead of a primerbinding region, a self-priming template has a "self-priming region".This self-priming region contains two intra-complementary regionsseparated by several, usually 3 to 5, nucleotides. Theintra-complementary regions form a duplex, and the several nucleotidesseparating the intra-complementary regions form a loop. The 3' terminalbase must be paired, and the region of complementarity must besufficient to allow priming to occur. The resultant structure isreferred to as a "hairpin". (Dattagupta, European Patent Application No.427,073A2.) See FIG. 4. As shown, the 3'-terminus of the self-primingtemplate serves as the primer.

It is also possible to design templates with primer complementaryregions such that the primer complementary region of one templatemolecule will hybridize with the primer complementary region of anothertemplate molecule to form a duplexed primer complementary region. Inthis manner, one template serves as the primer for another, thuseliminating the need for a separate primer. These templates can bereferred to as "primer/templates", since they serve both functions. SeeFIG. 5. As shown, using DNA primer/templates with a single3'-ribonucleotide, the primer/template can be separated from theoligonucleotide product via alkaline hydrolysis and/or RNase.

The Primer

The primer can be synthesized using any of the aforementioned methodsfor synthesizing template. The primer can be either DNA or RNA, or amodified DNA or RNA. A particularly useful primer is a 3'-ribonucleotideprimer which consists of DNA with several, preferably 1 to 4,ribonucleotides at or near (i.e. within 1 to 3 nucleotides of) its3'-terminus. Preferably, the primer is a 3'-ribonucleotide primerconsisting of DNA with a single ribonucleotide residue at its3'-terminus. This type of primer has been utilized to preventcontamination during PCR (Walder et al., European Patent Application496,483 A2). As used herein, 3'-ribonucleotide primers provide aconvenient cleavage site for separation of the primer from theoligonucleotide product, since the internucleotide linkage which is 3'to a ribonucleotide residue is cleavable by chemical or enzymatic means.

The 3'-ribonucleotide primers are preferred, because they are capable ofretaining their function after cleavage of the primer extension productto separate the primer from the oligonucleotide product. A primer which"retains its function" after cleavage is one which is capable ofremaining hybridized to the same template, or hybridizing to a differenttemplate, to initiate a subsequent synthesis reaction.

Ribonucleotide-containing primers can be prepared using known methods ofoligonucleotide synthesis. For example, see Walder, et al., supra. Whena ribonucleotide-containing primer is used with an RNA template,cleavage of the primer extension product and digestion of the templatecan be accomplished simultaneously. See FIG. 6. See below for a morecomplete description of primer extension product cleavage and templatedigestion.

Another useful primer consists of an oligonucleotide which contains atleast one deoxyuridine at the 3'-terminus. After synthesis of the primerextension product, the deoxyuridine residue can be excised by thecombined action of a DNA glycosylase, followed by cleavage of thephosphate bond by an apurine/apyrimidine endonuclease (AP-endonuclease),and removal of the resultant exposed baseless sugar by an exonuclease.(See McGilvery, et al., Biochemistry: A functional Approach, W. B.Saunders Co., Philadelphia, Pa., 125-126 (1983).)

Formation of the Template-Primer Hybrid

Prior to oligonucleotide synthesis, a primer must be hybridized to thetemplate. A template and primer combination should be used such that theprimer binds to the primer binding region and not to any other region ofthe template. Thus, primers should be long enough and sufficientlycomplementary to the primer complementary region to form a stable duplexwith it. The duplex thus formed allows the 3'-OH terminus of the primerto function as an initiation site for synthesis.

The primer and the template can be prehybridized prior to use, but arepreferably added sequentially or simultaneously to the synthesisreaction mixture prior to addition of the polymerase. The hybridizationconditions necessary to cause specific duplex formation between theprimer and the template will most importantly depend on the specificityof the primer for the template's primer binding region, but will alsodepend to some degree on the heterogeneity of the nucleic acids present.The hybridization conditions must also be compatible with the polymeraseenzyme being able to perform efficient and accurate synthesis. Ifchemically produced template is used wherein contamination by exogenousnucleic acids would be expected to be minimal, a wide range ofhybridization conditions can be used to selectively hybridize the primerto the template. When the template is produced enzymatically in thepresence of contaminating nucleic acids, hybridization conditions mustbe more stringent in order for selective hybridization of the primer andthe template to occur. Stringency can be increased by any known method,such as lowering ionic strength, increasing washing temperatures, and/orusing a denaturing agent.

Formation of the Prime Extension Product

The 3'-OH terminus of the primer in the primer-template hybrid serves asthe initiation site for synthesis via sequential addition of dNTPs inthe 5' to 3' direction. Alternatively, when a self-priming template isutilized, it is the 3'-OH terminus of the template's self-complementaryregion which serves as the initiation site for synthesis. The mixture ofnucleotides to be used in the synthesis may contain either allunmodified, all modified, or both unmodified and modified dNTPs inappropriate ratios, such that a desired amount of incorporation of themodified dNTP is achieved. If a fully phosphorothioated oligonucleotideis to be synthesized, only dNTPαSs will be used. The modified dNTPs usedmay be the same or different, but are preferably the same (i.e. allmethylphosphonates, all phosphorothioates, etc.) In addition, themixture of dNTPs which are available for synthesis must contain all ofthe nucleotides necessary for formation of the primer extension product.

The choice of polymerase will depend on the template and the substratesused, and any requirements for specific reaction conditions (e.g.thermostable polymerases for high temperature reactions). Polymerasessuitable for use with a variety of templates and substrates are wellknown in the art.

DNA-dependent DNA polymerases synthesize a complementary DNA copy of aDNA template. Examples of suitable DNA-dependent DNA polymerasesinclude, but are not limited to: DNA polymerase I (Burgess, et al.,Journal of Biol. Chem. 254: 6889-6893 (1979)); T4 polymerase (Romaniuk,et al., Journal of Biol. Chem. 257: 7684-7688 (1982)); and T7 polymerase(Brody, et al., Biochemistry 21: 2570 (1982)). Each of these polymerasescan also be used to synthesize phosphorothioate oligonucleotide. Allknown DNA-dependent DNA polymerases require a complementary primer toinitiate synthesis. Generally, the primer may be RNA or DNA, or acopolymer of RNA and DNA, to initiate synthesis. It is known that undersuitable conditions certain DNA-dependent DNA polymerases may alsosynthesize a complementary DNA copy of an RNA template.

RNA-dependent DNA polymerases, also called reverse transcriptases,synthesize a complementary DNA copy of an RNA template. Examples ofsuitable RNA-dependent DNA polymerases include, but are not limited toavian myeloblastosis virus reverse transcriptase (Bartlett, et al.,Journal of Biol. Chem. 257 (15): 8879-8854 (1982)). This enzyme, as wellas other reverse transcriptases known in the art, is also capable ofbeing used to synthesize phosphorothioate oligonucleotides. All knownreverse transcriptases also have the ability to make a complementary DNAcopy of a DNA template and are thus both RNA- and DNA-dependent DNApolymerases.

Cleavage of the Primer Extension Product

The primer extension product will necessarily have at least one cleavagesite, and may have several equivalent or different cleavage sites. Onecleavage site is necessary to separate the oligonucleotide product fromthe primer and, if the template's oligonucleotide complementary regionconsist of two or more subregions each having a sequence complementaryto an individual oligonucleotide, the oligonucleotide product mustcontain cleavage sites for separation of the individualoligonucleotides.

When using a template to synthesize more than one individualoligonucleotide, it may be desirable to form a primer extension productwith more than one type of cleavage site, one of which may be thephosphate linkage adjacent to a ribonucleotide residue which was formedby using a 3'-ribonucleotide primer, and the other of which may be arestriction endonuclease recognition sequence. In this manner, theprimer extension product can be cleaved once to separate the primer, andthen cleaved again (before or after purification) to separate theindividual oligonucleotides from each other. See FIG. 7.

Different restriction sites can be incorporated into the primerextension product, each of which is recognized by a differentrestriction endonuclease, such as those identified in Example II below.An important aspect of the present invention is the discovery of severalpreviously unknown restriction endonuclease/restriction sequencecombinations which result in the cleavage of phosphorothioateoligonucleotides.

That these restriction sequences were capable of being recognized inphosphorothioate oligonucleotides and cleaved was an unexpected finding.Taylor, et al. (Nucleic Acids Research, 13(24): 8749-8764 (1985)) havereported that several restriction endonucleases, such as Alu I, arecapable of cleaving partially phosphorothioated oligonucleotides. It wasexpected that there would be a correlation between the ability ofrestriction endonucleases to cleave partially phosphorothioatedoligonucleotides and their ability to cleave fully phosphorothioatedoligonucleotides. However, this was found not to be the case in allinstances, and the efficiency of a restriction endonuclease to cleavefully phosphorothioated oligonucleotides could not be predicted based onprevious results with partially phosphorothioated oligonucleotides. SeeExamples I to III.

In addition to the use of site-specific restriction endonucleases, if a3'-ribonucleotide primer was used in conjunction with a DNA template,cleavage to separate the primer and the oligonucleotide product can beaccomplished with alkaline hydrolysis, or by the use of an endonuclease,such as RNase.

RNase H's may contain either or both endonuclease or exonucleaseactivity. Arian myeloblastosis virus and Moloney murine leukemia virusreverse transcriptases contain an RNase H activity in addition to theirpolymerase activity. However, some cloned reverse transcriptases lackRNase H activity. There are also sources of RNase H available without anassociated polymerase activity. Although the cleavage site of most RNaseH's requires the presence of more than one ribonucleotide residue, RNaseH's which are capable of cleaving a DNA strand containing a singleinternal ribonucleotide residue have been reported. (See Walder, et al.,supra).

Cleavage of a primer extension product which is formed using a3'-ribonucleotide primer occurs in the phosphate linkage(s) which is/arein the 3' direction from the ribonucleotide residue(s). Cleavage byalkali causes the formation of a mixture of 2'-, 3'- and 2',3'-cyclicphosphate ribonucleotides, which can then be "regenerated" by convertingit back to a 3'-OH by alkaline phosphatase. Preferably, an RNase is usedwhich results in reformation of a primer with a 3'-OH terminus. When the3'-ribonucleotide primer consists of DNA with a single ribonucleotideresidue at the 3' terminus, the regenerated or reformed primer will beidentical to the original primer. Such primers are particularlypreferred for this reason.

If a deoxyuridine-containing primer is used, cleavage can beaccomplished by hydrolysis with a DNA glycosylase, followed by cleavageof the phosphate bond by an apurinic/apyrimidinic-endonuclease("AP-endonuclease") and removal of the baseless sugar by an exonuclease.(See McGilvery, et al., Biochemistry: A functional Approach, supra.)

In instances where the primer and the oligonucleotide to be synthesizedare both the same type of nucleic acid, for example both unmodified RNAor DNA, it is also possible for some of the nucleotides of the primer tobe incorporated into the oligonucleotide product after cleavage of theprimer extension product. In this situation, cleavage of the primerextension product occurs in the 5' direction from the original3'-terminus of the primer, and one or more of the primer's nucleotidesbecome incorporated into the 5' end of the oligonucleotide.Alternatively, it is possible for cleavage of the primer extensionproduct to occur in the 3' direction from the original 3'-terminus ofthe primer. However, in both instances, the primer will not be able tobe used in subsequent synthesis reactions.

In instances where a DNA primer/template with a single 3'-ribonucleotideis used, cleavage at the ribonucleotide residue as described above,followed by separation and purification of the oligonucleotide product,can result in a fully regenerated and reusable primer/template.

Digestion of the Template

The template is preferably capable of being digested after formation ofthe primer extension product. Using nucleases which will only recognizethe double-stranded product formed after synthesis of theoligonucleotide, it is possible to digest the template only afterformation of the primer extension product without digestingsingle-stranded template that has not yet directed the synthesis of aprimer extension product. Digestion of the template can occur eitherbefore, simultaneously with or after cleavage of the primer extensionproduct. Digestion of the template can serve two useful purposes; (1)synthesis reaction components can easily be recovered and reused; and(2) digestion leaves the oligonucleotide and the primer (or in the caseof pre-cleavage digestion, the primer-extension product) insingle-stranded form thus facilitating purification and/or separationfrom the synthesis reaction mixture.

If the template is DNA, digestion can be accomplished using a DNase. Theoligonucleotide product can be protected from nuclease digestion byvirtue of the incorporation of modified linkages during synthesis. If amodified primer is also used which is not digested by the DNase, thetemplate can be selectively digested in the presence of primer,oligonucleotide and/or primer-extension product.

Preferably, the template is RNA, in which case digestion can easily beaccomplished using alkaline hydrolysis and/or a suitable RNase.

EXAMPLE I Determination of Restriction Endonuclease Effectiveness

The primer extension product can be cleaved with restrictionendonucleases, provided that the template is designed to direct thesynthesis of a primer extension product that contains a sequence whichcan be recognized by such an enzyme. Many restriction endonucleaserecognition sequences are known in the art. (See The Biochemistry of theNucleic Acids: Chapter 4, Degradation and Modification of Nucleic Acids,supra.)

Due to the resistance of many modified oligonucleotides to be recognizedand thus cleaved by restriction endonucleases, it is necessary toprescreen restriction endonucleases prior to use in oligonucleotidesynthesis for their ability to cleave a nucleic acid which contains thesame modifications which are intended to be incorporated into the primerextension product. A screening method for phosphorothioateoligonucleotides follows.

Screening for restriction endonuclease activity was carried outaccording to the method described by Taylor, et al. (Nucleic AcidsResearch, 13(24): 8749-8764 (1985)). The substrate utilized wasdouble-stranded M13 DNA with a completely native phosphodiester-linked(+) strand and a completely phosphorothioate-linked (-) strand. Acontrol was prepared which contained DNA with native phosphodiesterlinkages in both strands. Each restriction endonuclease was tested forits ability to digest the substrate by incubating twenty units of eachrestriction endonuclease with 0.5 μg M13 DNA for 1 hour at thetemperature and under the conditions described by the enzymemanufacturer for native phosphodiester DNA.

The restriction fragments which were formed using the assay substratewere compared to those formed using the control, and results were scoredaccordingly. These results are reported in Table 1. A result reported as"++" indicates that complete cleavage of both the control and testsubstrates was obtained using the specified reaction conditions. Aresult reported as + indicates that the restriction endonuclease wascapable of cleaving the substrate, but less than complete cleavageoccurred (i.e. "partial cleavage"). Even a result of only partialcleavage using this assay indicates that the restriction endonucleaseshould be suitable for use in the synthesis ofphosphorothioate-containing oligonucleotides, since the cleavagereaction can easily be further optimized. An "effective amount" ofrestriction endonuclease is the amount necessary to effect cleavageunder specified conditions.

                  TABLE 1                                                         ______________________________________                                        RESTRICTION ENDONUCLEASE STUDY USING DOUBLE                                   STRANDED SUBSTRATE                                                            Enzyme*   Results       Recognition Sequence                                  ______________________________________                                        AlwN I    +             5'-CAGNNN/CTG-3'                                      BsaJ I    ++            5'-C/CNNGG-3'                                         Bsr I     ++            5'-ACTGGN/-3'                                         BstN I    ++            5'-CC/(A,T)GG-3'                                      Cla I     ++            5'-AT/CGAT-3'                                         Hae III   ++            5'-GG/CC-3'                                           HinP I    ++            5'-G/CGC-3'                                           Msp I     +             5'-C/CGG-3'                                           Mva I     ++            5'-CC/(A,T)GG-3'                                      ScrF I    ++            5'-CC/(N)GG-3'                                        Taqα I                                                                            ++            5'-T/CGA-3'                                           ______________________________________                                         *All endonucleases except for Mva I were obtained from New England            Biolabs, Inc., Beverly, MA. Mva I was obtained from Boehringer Mannheim,      Indianapolis, IN.                                                             N = A, T, C or G                                                              / = Location of cleavage                                                      (A,T) = Either nucleotide                                                

EXAMPLE II Optimization of Restriction Endonuclease Conditions

The effects of reaction conditions on cleavage efficiency of severalendonucleases were studied using the procedure described in Example I.In particular, the effects of substituting sodium acetate for NaCl, andMnCl₂ for MgCl₂ were studied in order to find conditions which favorcleavage. A similar optimization could be performed with any restrictionendonuclease by using the conditions reported as optimal by the enzymemanufacturer and varying the type and amount of cations, anions,buffers, etc.

Each of the NaCl and MgCl₂ concentrations were kept at standardconcentrations while varying the concentration of the other. Optimumsfor each are reported in Table 2 below. Then, while using the optimumconcentration of NaCl, a varying amount of MnCl₂ was added as asubstitute for MgCl₂. Likewise, while using the optimum concentrationMgCl₂, a varying amount of sodium acetate was added as a substitute forNaCl. The ability of MnCl₂ and sodium acetate to substitute for MgCl₂and NaCl, respectively, are also reported in Table 2.

                  TABLE 2                                                         ______________________________________                                        EFFECTS OF DIFFERENT REACTION CONDITIONS                                                               Sodium                                                                        acetate,       MnCl.sub.2,                                          NaCl, mM  mM     MgCl.sub.2, mM                                                                        mM                                    Enzyme                                                                              Conditions                                                                             (0 to 400)                                                                              (0 to 400)                                                                           (0.1 to 20)                                                                           (0.1 to 10)                           ______________________________________                                        Hae III                                                                             Standard 50     mM          10   mM                                           Optimum  0      mM   No effect                                                                            2-5  mM   No effect                         Taq.sup.α I                                                                   Standard 100    mM          10   mM                                           Optimum  50     mM   No effect                                                                            1-2  mM   0.5-2 mM                          HinP I                                                                              Standard 50     mM          10   mM                                           Optimum  50     mM   No effect                                                                            10   mM      2 mM                           BstN I                                                                              Standard 50     mM          10   mM                                           Optimum  0      mM   No effect                                                                            No   effect                                                                             No effect                         Mva I Standard 25     mM          10   mM                                           Optimum  100    mM   No effect                                                                            No   effect                                                                             No effect                         ScrF I                                                                              Standard 100    mM          5    mM                                           Optimum  0-50   mM   0-50 mM                                                                              5-10 mM      1 mM                           ______________________________________                                    

These results demonstrate that in most instances lowering theconcentration of sodium chloride tended to increase the rate ofphosphorothioate DNA cleavage. Similarly, lowering the magnesiumchloride concentration was generally favorable to cleavage. Only in afew instances was any effect on cleavage on observed by using MnCl₂instead of MgCl₂, or sodium acetate instead of NaCl.

EXAMPLE III Restriction Endonuclease Study using Single-StrandedSubstrate

As shown in FIG. 5, it is possible to use a ribose-containing primer incombination with an RNA template to synthesize multiple oligonucleotidesfrom a single template. Using alkali and/or RNase to cleave the primerextension product at the location of the ribose residue and to digestthe template, a single-stranded product remains which must be cleaved.In order to test the ability of the restriction endonucleases fromExample 1 to cleave a single-stranded target, an oligonucleotidesubstrate was chemically synthesized which contained restrictionendonuclease recognition sequences for all of the enzymes to be tested.The sequence of this oligonucleotide is given in SEQ. ID. NO. 1 Usingthe standard conditions reported in Example 2, several enzymes weretested for their ability to cleave the single-stranded oligonucleotidesubstrate. The results are given below in Table 3:

                  TABLE 3                                                         ______________________________________                                        RESTRICTION ENDONUCLEASE STUDY USING                                          SINGLE-STRANDED SUBSTRATE                                                             Enzyme                                                                              Results                                                         ______________________________________                                                BstN I                                                                              -                                                                       Hae III                                                                             +                                                                       HinP I                                                                              +                                                                       Mva I -                                                                       ScrF I                                                                              ++                                                                      Taq.sup.α I                                                                   +                                                               ______________________________________                                    

Results reported as "+" indicate partial cleavage of the oligonucleotidesubstrate. Results reported as "++" indicate complete cleavage of theoligonucleotide substrate. Results reported as "-" indicate nosignificant cleavage. Under these cleavage conditions, either resultindicates that the particular restriction endonuclease tested would besuitable for use.

EXAMPLE IV Synthesis of an Oligonucleotide from an RNA Template

A. Preparation Of Template

A template was chosen which would direct the synthesis of an antisenseoligonucleotide which was complementary to the mRNA sequence given bySEQ. ID. NO. 2 encoding the HIV REV protein. (See Peterson, et al.,Published PCT Application No. WO 95/03407.) Template was prepared byfirst chemically synthesizing an oligodeoxynucleotide having twosubsequences; a T7 RNA polymerase self-complementary ("hairpin")promotor sequence (see SEQ. ID. NO. 3), and a sequence (see SEQ. ID. No.4) which was complementary to the desired template. The sequence of thisoligodeoxynucleotide is given by SEQ. ID No. 5.

The in vitro transcription from the oligodeoxynucleotide was carried outin a 20 ml reaction mixture containing 0.256 A₂₆₀ units of theoligodeoxynucleotide in 40 mM Tris (pH 8.0), 20 mM MgCl₂, 4 mM ATP, 4 mMGTP, 4 mM CTP, 4 mM UTP, 1 mM spermidine, 0.08% (w/v) polyethyleneglycol (PEG) 8000, 0.01% (w/v) Triton X-100, 5 mM dithiothreitol (DTT),50 μg/ml bovine serum albumin (BSA), 800 units of RNasin (Promega, Inc.,Madison, Wis.), and 120,000 units of T7 RNA polymerase. The reactionmixture was incubated at 37° C. for approximately four hours, or untilformation of a magnesium pyrophosphate precipitate was observed.

The template thus formed had the sequence given by SEQ. ID. NO. 6, andconsisted of two subsequences; the primer binding region (SEQ. ID. NO.7) and the oligonucleotide complementary region (SEQ. ID. NO. 8).

The template was isolated by chromatography as follows: The magnesiumpyrophosphate precipitate was dissolved by the addition of EDTA to afinal concentration of 20 mM. The reaction mixture was concentrated byprecipitation with ethanol and redissolved in 10 ml of 100 mMtriethylammonium acetate, pH 7.5. The redissolved RNA was isolated byreverse-phase HPLC on a C₄ column eluted with a gradient ofacetonitrile. Fractions containing the RNA product were concentrated byethanol precipitation.

B. Synthesis of the Oligonucleotide Product

A DNA primer (with a 3' terminus consisting of UC, where U is aribonucleotide, and C is a deoxyribonucleotide) was used which had thesequence given by SEQ. ID. NO. 9. A 9 mL reaction mixture was preparedto contain 130 A₂₆₀ units of primer, 130 A₂₆₀ units of template, 50 mMTris, pH 8.3, 75 mM KCl, 25 mM NaF, 3 mM MgCl2, 1 mM DTT, 100 μg/mL BSA,200 units/ml RNasin, 1 mM dTTPαS, 1 mM dCTPαS and 1 mM dGTPαS (note that1 mM dATPαS was not required because the desired oligonucleotideconsisted only of C, G and T). Then, 3.6×10⁶ units of Moloney murineleukemia virus (MMLV) reverse transcriptase was added, and the reactionmixture was incubated for twenty one hours at 37° C.

C. Purification of the Oligonucleotide Product

The resulting double stranded product consisting of the primer extensionproduct (SEQ. ID. NO. 10) hybridized to the template was purified byextraction with toluene:phenol (9:1 v/v). The toluene:phenol phase wasextracted three times with 10 mM Tris (pH 8.0), and the combined aqueousphases were concentrated by precipitation with ethanol. The ethanolprecipitate was redissolved in 1.2 mL 10 mM Tris (pH 8.0), adjusted to100 mM NaOH, then incubated for 60 minutes at 70° C. This alkalinehydrolysis had a dual function: 1) separating the oligonucleotideproduct from the primer by cleavage of the phosphodiester bond betweenthe uridine of the primer and the cytidine of the oligonucleotide; and2) digesting the RNA template.

The alkaline-treated material was neutralized with HCl, then the RNAtemplate was treated with 12.5 μg E. coli RNase A for two hours toachieve more complete digestion of the RNA template. The oligonucleotideproduct (SEQ. ID. NO. 11) was concentrated by precipitation withethanol, redissolved in 100 mM triethylammonium acetate, pH 7.5, andisolated by high pressure liquid chromatography on a C₁₈ reverse phasecolumn eluted with acetonitrile using a 0.5% gradient. Peak fractions ofthe oligonucleotide product were pooled and concentrated by ethanolprecipitation, redissolved in sterile phosphate buffered saline, andstored at -20° C.

D. Regeneration and Reuse of the Primer

The "used" primer from step C was reused in a subsequent synthesisaccording to steps A to C above and found to be 28% as active as anunused primer. Treatment of the used primer with calf intestine alkalinephosphatase resulted in the complete regeneration of the primer, asdemonstrated by its reuse in a subsequent synthesis according to steps Ato C with 100% the activity of an unused primer.

EXAMPLE V Comparison of Oligonucleotide Product to ChemicallySynthesized Oligonucleotide

In order to compare chemically synthesized oligonucleotides toenzymatically synthesized oligonucleotides, a phosphorothioateoligonucleotide was prepared using automated chemical synthesis (the"control") to have the same sequence as the oligonucleotide product fromExample IV (the "product").

A. Comparison of Physiochemical Properties

The product appeared as a single band on a 20% denaturing polyacrylamidegel and had an electrophoretic mobility substantially identical to thatof the control oligonucleotide. Additionally, the thermal denaturationcurves of the product and the control were identical.

The product and the control were also compared using nuclear magneticresonance spectroscopy calibrated with H₃ PO₄. The signal obtained byboth exhibited a peak at 55.639 ppm, and the product exhibited anupfield chemical shift which was consistent with the product containingphosphorothioate linkages with the Rp configuration.

B. Comparative Effect on p24 Production

The product was tested for effectiveness as an antisense agent in vitroand compared to the control. T-lymphocytes (SupT-1 from AdvancedBioTechnologies, inc., Columbia, Md.) were propagated in RPMI 1640medium supplemented with 10% (v/v) fetal bovine serum and 50 μg/mlgentamicin sulfate at 37° C. in a humidified 5% CO₂ atmosphere. Onlycell cultures having viable titers less than 2×10⁶ cells/ml andviability in excess of 90%, as gauged by trypan blue exclusion, wereused as hosts for acute infection.

Approximately 2.0×10⁶ cells were pelleted by centrifugation at about 170g for 8 minutes. The medium was removed and the cells were gentlyresuspended in fresh medium to a final concentration of about 1×10⁶cells/ml. HIV-1 strain IIIB (1×10⁵ TCID₅₀ /ml; "TCID"=Tissue CultureInfective Dose), was added to the cells at a multiplicity of infectionof 0.04 syncytium-forming units ("sfu") per cell (0.7 sfu=1.0 TCID₅₀).The virus and cell mixture was incubated for 2 hours at 37° C. in ahumidified 5% CO₂ atmosphere, and then diluted to 10 ml with medium andpelleted by centrifugation at about 170 g for 8 minutes. The pelletedcells were washed three times with 10 ml of medium and then resuspendedin medium to a concentration of 1×10⁵ cells/ml.

Cells were dispensed in 100 μl volumes to round bottom wells of 96-wellplates containing an equal volume of medium with various concentrationsof product or control oligonucleotide, as well as a "no oligonucleotide"blank. Each concentration was tested at least twice. The plates wereincubated at 37° C. in a humidified 5% CO₂ atmosphere. After 7 days, thecells in each culture were pelleted in situ by centrifuging theincubation plate at about 170 g for 8 minutes. One hundred μl of thesupernatant from each well was transferred to a new 96-well plate andfrozen at -80° C. for later p24 core antigen level determination.

The supernatants were allowed to thaw at room temperature and diluted tovarious levels in fresh medium. HIV-1 p24 antigen levels were determinedusing a capture ELISA purchased from Coulter Corporation (Hialeah,Fla.). The kinetic assay format was used and carried out according tothe manufacturer's instructions. The effect on p24 antigen productionwas virtually the same between the product and the control.

C. Comparative Effect on Formation of Syncitia

The effect of the product and the control on plaque formation wasdetermined as follows: HT-6C cells (clone 6C of Hela cells expressingCD4 from a recombinant retroviral vector, NIH AIDS Research andReference Reagent Program) were maintained in 75 cm² tissue cultureflasks in DMEM medium (Gibco BRL, Gaithersberg, Md.) supplemented with10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin,2 mM glutamine at 37° C. in a humidified 5% CO₂ atmosphere. The cellswere detached from the flasks with trypsin-EDTA (GibcoBRL, Gaithersberg,Md.), collected by centrifugation at 230 g and resuspended in the abovemedium. These cells were plated at 3.2×10⁴ cells/well in 48-well tissueculture dishes and grown overnight at 37° C. in a humidified 5% CO₂atmosphere.

To initiate an assay, the medium was removed from each well and 200 μlof HIV (100 to 200 plaque forming units) in DMEM medium supplementedwith 4% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 μg/mlstreptomycin, 2 mM glutamine, 8 μg/ml DEAE dextran, and 0.5 μg/mlpolybrene were added to each well. The dishes were incubated at 37° C.in a humidified 5% CO₂ atmosphere. After 2 hours, 800 μl of mediumcontaining various concentrations of product or control was added towells, and the dishes were each incubated at 37° C. in a humidified 5%CO₂ atmosphere for three days. The medium from each well was thenremoved and 1 ml of 100% methanol was added to each well to fix thecells to the dishes. After 15 minutes, the methanol was removed and 0.5ml of 0.3% (w/v) crystal violet stain dissolved in phosphate bufferedsaline was added to each well. After 5 minutes, the wells were rinsedwith water, drained and allowed to dry. The number of syncitia (darkstaining giant cells) in each well were counted using microscopicexamination. The percent reduction in syncitium formation resulting fromtreatment with various concentrations of the oligonucleotide product andthe control oligonucleotide were virtually the same.

EXAMPLE VI Synthesis of an Oligonucleotide using a Self-Priming RNATemplate

A. Preparation of Template

A template was chosen which would direct the synthesis of an antisenseoligonucleotide which was complementary to an mRNA sequence (SEQ. ID No.2) encoding the HIV REV protein. (See Peterson, et al., supra.) Templatewas prepared by first chemically synthesizing an oligodeoxynucleotidehaving three subsequences given in 5' to 3' order: a T7 RNA polymeraseself-complementary ("hairpin") promotor sequence (SEQ. ID. NO. 12); anda sequence which was complementary to the desired self-priming template(SEQ. ID. NO. 13). The sequence of this oligodeoxynucleotide, whichterminated in a 5' non-nucleotide "RXL" linker, is given by SEQ. ID No.14. (For the precise structure of the RXL linker, see Arnold, et al.,PCT WO 89/02439, linker reagent 23 of Example 8(a), incorporated hereinby reference).

The in vitro transcription from the oligodeoxynucleotide was carried outin a 2.0 ml reaction mixture containing 0.252 A₂₆₀ units of theoligodeoxynucleotide in 40 mM Tris (pH 8.0), 20 mM MgCl₂, 4 mM ATP, 4 mMGTP, 4 mM CTP, 4 mM UTP, 1 mM spermidine, 0.08% (w/v) polyethyleneglycol (PEG) 8000, 0.01% (w/v) Triton X-100, 5 mM dithiothreitol (DTT),5 μg/mL bovine serum albumin (BSA), 100 units of RNasin (Promega, Inc.,Madison, Wis.), and 24,000 units of T7 RNA polymerase. The reactionmixture was incubated at 37° C. for approximately four hours, or untilformation of a magnesium pyrophosphate precipitate was observed.

The self-priming template thus formed had the sequence given by SEQ. ID.NO. 15, and consisted of two subsequences in 5' to 3' order; theoligonucleotide complementary region (SEQ. ID. NO. 16) and theself-priming region (SEQ. ID. NO. 17).

The template was isolated by chromatography as follows: The magnesiumpyrophosphate precipitate was dissolved by the addition of EDTA to afinal concentration of 20 mM. The reaction mixture was concentrated withethanol, and redissolved in 0.1M triethylammonium acetate, pH 7.5. Theredissolved RNA was applied to a Sep-Pak C₁₈ cartridge (MilliporeWaters, Milford, Mass.) and washed with 0.1M triethylammonium acetate,pH 7.5. The template was eluted with 70% methanol in water, concentratedby vacuum evaporation and redissolved in 0.1M triethylammonium acetate,pH 7.5. The RNA transcript was further purified by high pressure liquidchromatography.

B. Synthesis of Oligonucleotide Product

A 1 mL reaction mixture was prepared to contain 7.45 A₂₆₀ units ofself-priming template, 50 mM Tris, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 336units/ml RNasin, 3 mM dTTPαS, 3 mM dCTPαS and 3 mM dGTPαS (note that 1mM dATPαS was not required because the desired oligonucleotide consistedonly of C, G and T). Then; 1.3×10⁶ units of Moloney murine leukemiavirus (MMLV) reverse transcriptase was added, and the reaction mixturewas incubated for seven hours at 37° C.

EXAMPLE VII Synthesis of a Plurality of Oligonucleotides from a SingleTemplate

A. Preparation of Template

A template was chosen which would direct the synthesis of an antisenseoligonucleotide which was complementary to an mRNA sequence (SEQ. ID No.18) encoding the HIV REV protein. (See Peterson, et al., supra.)Template was prepared by first chemically synthesizing anoligodeoxynucleotide having two subsequences; a T7 RNA polymerasepromotor sequence (SEQ. ID. No. 19), and a sequence (SEQ. ID. No. 20)which was complementary to the desired template, and which consisted ofthree repeated subsequences. The sequence of this oligodeoxynucleotideis given by SEQ. ID No. 21.

The in vitro transcription from the oligodeoxynucleotide was carried outin a 0.1 ml reaction mixture containing 0.003 A₂₆₀ units of theoligodeoxynucleotide in 40 mM Tris (pH 8.0), 20 mM MgCl2, 4 mM ATP, 4 mMGTP, 4 mM CTP, 4 mM UTP, 1 mM spermidine, 0.08% (w/v) polyethyleneglycol (PEG) 8000, 0.01% (w/v) Triton X-100, 5 mM dithiothreitol (DTT),50 μg/mL bovine serum albumin (BSA), and 600 units of T7 RNA polymerase.The reaction mixture was incubated at 37° C. for three hours. Thereaction was stopped by heating for 10 minutes at 70° C. After coolingto 0° C., 80 units of RNasin (Promega, Inc., Madison, Wis.) were added.

The template thus formed had the sequence given by SEQ. ID. NO. 22.

B. Synthesis of Oligonucleotide Product

The template was reverse transcribed without further purification. A 10μl aliquot of the reaction mixture was added to a synthesis reactionmixture containing 19 ng dT₁₀ primer, 50 mM Tris HCl (pH 8.3), 75 mMKCl, 3 mM MgCl2, 2 mM dATPαS, 2 mM dTTPαS, 2 mM dGTPαS, 2 mM dCTPαS, and80 units of RNasin. To this synthesis reaction mixture was added RNase Hfree MMLV reverse transcriptase (Gibco BRL, Bethesda, Md.) to a totalvolume of 100 μl. This oligonucleotide product precursor thus formed waspurified on a NENSORB 20 cartridge (DuPont NEN, Boston, Mass.) accordingto manufacturer's instructions. This was followed by cleavage intoindividual oligonucleotides by treatment with 35 units of Hae III (NewEngland Biolabs, Beverly, Mass.) and 1 μg RNase (DNase-free,Boehringer-Mannheim, Indianapolis, Ind.) in Restriction Buffer 2 (NewEngland Biolabs, Beverly, Mass.) for 22 hours at 37° C. The sequence ofthe oligonucleotide product is given in SEQ. ID. NO. 23.

Although the invention has been described in terms of specificembodiments, many modifications and variations of the present inventionare possible in light of the teachings. It is, therefore, to beunderstood that within the scope of the appended claims the inventionmay be practiced otherwise than as specifically described.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 23                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAAGCCCGGACAGCCCAGGGGAGCCCGGCCAGGCGCTCGAGAAGCCCGGACAGCCCAGGG60                GAGCCCTCGCCTATTGTTAAAGTGTGTCCT90                                              (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CCCGAGGGGACCCGACAGGCCCGAAG26                                                  (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TATAGTGAGTCGTATTATTTTTAATACGACTCACTATA38                                      (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GACGGCACCTCTTCGGGCCTGTCGGGTCCCCTCGGGCCC39                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 77 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GACGGCACCTCTTCGGGCCTGTCGGGTCCCCTCGGGCCCTATAGTGAGTCGTATTATTTT60                TAATACGACTCACTATA77                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GGGCCCGAGGGGACCCGACAGGCCCGAAGAGGTGCCGTC39                                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AGGTGCCGTC10                                                                  (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GGGCCCGAGGGGACCCGACAGGCCCGAAG29                                               (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GACGGCACCUC11                                                                 (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GACGGCACCTCTTCGGGCCTGTCGGGTCCCCTCGGGCCC39                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CTTCGGGCCTGTCGGGTCCCCTCGGGCCC29                                               (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TATAGTGAGTCGTATTATTTTTAATACGACTCACTATAGC40                                    (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AAAAAAAAAAGCTTTCGTTTTTTTTTTCTTCGGGCCTGTCGGGTCCCCTCGGGGC55                     (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AAAAAAAAAAGCTTTCGTTTTTTTTTTCTTCGGGCCTGTCGGGTCCCCTCGGGGCTATAG60                TGAGTCGTATTATTTTTAATACGACTCACTATAGC95                                         (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GCCCCGAGGGGACCCGACAGGCCCGAAGAAAAAAAAAACGAAAGCTTTTTTTTTT55                     (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GCCCCGAGGGGACCCGACAGGCCCGAAG28                                                (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      AAAAAAAAAACGAAAGCUUUUUUUUUU27                                                 (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GGGACCCGACAGGC14                                                              (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TATAGTGAGTCGTATTATTTTTAATACGACTCACTATAGC40                                    (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TTTTTTTTTTGGCCGCCTGTCGGGTCCCGGCCGCCTGTCGGGTCCCGGCCGCCTGTCGGG60                TCCCGGCCGC70                                                                  (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 110 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      TTTTTTTTTTGGCCGCCTGTCGGGTCCCGGCCGCCTGTCGGGTCCCGGCCGCCTGTCGGG60                TCCCGGCCGCTATAGTGAGTCGTATTATTTTTAATACGACTCACTATAGC110                         (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GCGGCCGGGACCCGACAGGCGGCCGGGACCCGACAGGCGGCCGGGACCCGACAGGCGGCC60                (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CCGGGACCCGACAGGCGG18                                                          __________________________________________________________________________

We claim:
 1. A method of synthesizing at least one phosphorothioateoligonucleotide or phosphorothioate-containing oligonucleotidecomprising the steps of:(a) providing a nucleic acid template, saidtemplate comprising a primer binding region and an oligonucleotidecomplementary region located 5' to said primer binding region,whereinsaid oligonucleotide complementary region comprises one or moresubregions, each said subregion being complementary to an individualphosphorothioate or phosphorothioate-containing oligonucleotide to besynthesized, wherein said template is used to direct the synthesis of aprimer extension product comprising a nucleic acid primer, anoligonucleotide product comprising said oligonucleotides, and one ormore cleavage sites, said primer and said oligonucleotide product beingseparated by one of said cleavage sites and, when more than oneoligonucleotide is to be synthesized, each pair of adjacentoligonucleotides is separated by one of said cleavage sites, wherein atleast one of said cleavage sites is a restriction endonucleaserecognition sequence selected from the group consisting of 5'-GGCC-3',5'-GCGC-3', 5'-CCNGG-3', 5'-TCGA-3', and wherein N is any nucleotide andeach nucleotide base of said recognition sequence is phosphorothioated;(b) contacting said template with said primer under conditions such thatsaid primer hybridizes to the primer binding region of said template toform a template-primer hybrid; (c) incubating said template-primerhybrid in the presence of at least one DNA polymerase under conditionssuch that DNA synthesis occurs to form said primer extension product;and (d) cleaving said primer extension product such that said primer isseparated from said oligonucleotide product and the oligonucleotides ofsaid oligonucleotide product are separated from each other, wherein atleast one cleavage is accomplished by contacting the primer extensionproduct with an effective amount of a restriction endonuclease capableof recognizing said recognition sequence, said restriction endonucleasebeing selected from the group consisting of Hae III, HinP I, ScrF I andTaq.sup.α I.
 2. The method of claim 1, wherein said restrictionendonuclease recognition sequence is 5'-GGCC-3' and said restrictionendonuclease is Hae III.
 3. The method of claim 1, wherein saidrestriction endonuclease recognition sequence is 5'-GCGC-3' and saidrestriction endonuclease is HinP I.
 4. The method of claim 1, whereinsaid restriction endonuclease recognition sequence is 5'-CCNGG-3' andsaid restriction endonuclease is ScrF I.
 5. The method of claim 1,wherein said restriction endonuclease recognition sequence is 5'-TCGA-3'and said restriction endonuclease is Taq.sup.α I.
 6. The method of claim1, wherein said template is RNA.
 7. The method of claim 1, wherein saidtemplate is DNA.
 8. The method of claim 1, wherein said oligonucleotideproduct comprises two or more oligonucleotides having the same ordifferent lengths and/or sequences.
 9. The method of claim 1, whereinsaid primer comprises DNA and at least one ribonucleotide located at ornear the 3'-terminus of said primer.
 10. The method of claim 9, whereinsaid oligonucleotide product comprises two or more oligonucleotides andsaid primer is cleaved from said oligonucleotide product using alkalinehydrolysis.
 11. The method of claim 1, wherein said primer contains atleast one deoxyuridine located at or near its 3'-terminus.
 12. Themethod of claim 11, wherein said oligonucleotide product comprises twoor more oligonucleotides and said primer is cleaved from saidoligonucleotide product using a DNA glycosylase and an AP-endonuclease.13. The method of claim 1, wherein said template and said primer arecontained on the same nucleic acid strand, such that said template is aself-priming template comprising a self-complementary region and anoligonucleotide complementary region, and wherein said primer comprisesthe 3'-terminus of said self-complementary region.
 14. The method ofclaim 1, wherein said template is digested before, simultaneous with orsubsequent to step (d) using a alkaline hydrolysis.
 15. The method ofclaim 1, wherein said primer comprises the primer complementary regionof a second template.
 16. The method of claim 9, wherein saidoligonucleotide product comprises two or more oligonucleotides and saidprimer is cleaved from said oligonucleotide product using RNase Hactivity.
 17. The method of claim 1, wherein said template is digestedbefore, simultaneous with or subsequent to step (d) using an RNase Hactivity.
 18. The method of claim 1, wherein said cleavage sites can bethe same or different.
 19. The method of claim 1, wherein saidoligonucleotides are phosphorothioate oligonucleotides.
 20. The methodof claim 19, wherein said oligonucleotides are chirally pure.